Non‐Typeable Haemophilus influenzae Infection of the Junbo Mouse

Michael T. Cheeseman1, Derek W. Hood2

1 Developmental Biology Division, The Roslin Institute and Royal (Dick) School of Veterinary Studies University of Edinburgh, 2 Molecular Genetics Unit, MRC Harwell Institute, Harwell Science and Innovation Campus
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.24
Online Posting Date:  March, 2017
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Abstract

Acute otitis media, inflammation of the middle ear bulla, is the most common bacterial infection in children. For one of the principal otopathogens, non‐typeable Haemophilus influenzae (NTHi), animal models allow us to investigate host‐microbial interactions relevant to the onset and progression of infection and to study treatment of middle ear disease. We have established a robust model of NTHi middle ear infection in the Junbo mouse. Intranasal inoculation with NTHi produces high rates of bulla infection and high bacterial titers in bulla fluids; bacteria can also spread down the respiratory tract to the mouse lung. An innate immune response is detected in the bulla of Junbo mice following NTHi infection, and bacteria are maintained in some ears at least up to day 56 post‐inoculation. The Junbo/NTHi infection model facilitates studies on bacterial pathogenesis and antimicrobial intervention regimens and vaccines for better treatment and prevention of NTHi middle ear infection. © 2017 by John Wiley & Sons, Inc.

Keywords: Junbo mouse; lung infection; middle ear infection; non‐typeable Haemophilus influenzae (NTHi); otitis media

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Junbo Mouse NTHi Middle Ear Infection
  • Support Protocol 1: Junbo Mouse NTHi Middle Ear Infection: Histology and Distribution of NTHi
  • Support Protocol 2: Immune Response Measurement by Real‐Time Quantitative PCR (RT‐qPCR) of Bulla Fluids
  • Basic Protocol 2: Mouse Immunization and Protection Against NTHi Infection
  • Basic Protocol 3: Antimicrobial Treatment of NTHi Infection
  • Basic Protocol 4: NTHi Mouse Pulmonary Infection Model
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Junbo Mouse NTHi Middle Ear Infection

  Materials
  • NTHi [stored at –80ºC in BHI (see below)/20% (v/v) glycerol]: human OM disease isolates (162, 176, 375, 486, 1124 and 1158; available from MRC Harwell Institute) are used by us for mouse infection studies (Cody et al., ); streptomycin‐resistant NTHi strains are designated sr, e.g., strain 162sr
  • Supplemented brain‐heart infusion (sBHI) broth and agar plates (see recipe)
  • Mice (8 to 11 weeks of age): Junbo mice are congenic on a C3H/HeH background (Parkinson et al., ); mice are housed under SPF conditions and are mostly used at 8 to 11 typically used by us at 8±1 or 11±1 weeks of age with a similar experimental outcome [available from the European Mouse Mutant Archive (EM:00091) via MRC Harwell Institute; for non‐academic groups, available through MRC Technology, U.K.]; for some studies, germ‐free (GF) mice are used (details of the mouse husbandry and microbiological surveillance are found in Hood et al., )
  • Sterile PBS/2% (w/v) gelatin (Fluka, cat. no. 48723) for inoculum preparation
  • Barbiturate solution (50% Euthatal) for intraperitoneal injection (delivered at 3.3 ml/kg body weight); alternatively a rising CO 2 concentration (Donovan and Brown, ) may be used to euthanize mice
  • PBS for collecting and diluting in vivo samples
  • 70% ethanol to sterilize the instruments between the sampling of ears
  • 37ºC, 5% CO 2 incubator
  • 1‐μl inoculating loop
  • Spectrophotometer for determining OD 490
  • Scissors for removal of the head
  • Binocular dissecting microscope with 10× magnification and LED stage lighting.
  • Fine forceps for puncture of the tympanic membrane
  • < 2‐µl‐volume pipet with sterile filtered micro‐tip to collect the small volume of ME fluids
  • Additional reagents and equipment for assessing titer of bacteria (Elbing and Brent, ), isoflurane anesthesia of mice (Phoon and Turnbull, ), injection of mice (Donovan and Brown, ), and euthanasia of mice by CO 2 asphyxiation (Donovan and Brown, )

Support Protocol 1: Junbo Mouse NTHi Middle Ear Infection: Histology and Distribution of NTHi

  Materials
  • Dissected mouse heads ( protocol 1)
  • Xylene
  • 10% neutral buffered formalin
  • 100% ethanol
  • Tris‐buffered saline (TBS: see recipe)
  • Dako REAL peroxidase blocker (code no. S2023)
  • Dako proteinase K (code no. S3020)
  • Primary antibody [e.g., rat monoclonal anti‐F4/80 (Serotec, cat. no. MCA497G); rabbit polyclonal anti‐caspase 3 (Abcam, cat. no. ab2302); rabbit polyclonal anti‐histone 3 (Abcam, cat. no. ab61251)]
  • ImmPress HRP anti‐rat kit (Vector Labs, cat. no. MP‐744‐15)
  • Dako antibody diluent (code no. S0809)
  • Dako REAL peroxidase blocker (code no. S2023)
  • Dako liquid DAB+ substrate chromogen system (code no. K3468)
  • Harris hematoxylin (Sigma‐Aldrich, cat. no. HH516)
  • Dako Envision+ System HRP anti‐rabbit antibody (code no. K4011)
  • HRP Visualization Kit (Advanced Cell Diagnostics)
  • ClearVue mountant (Thermo Fisher Scientific)
  • Probe B‐HInfluenzae‐NTHi375‐16SrRNA (Advanced Cell Diagnostics)
  • Positive control for RNA integrity (e.g., PpiB; Advanced Cell Diagnostics)
  • Negative hybridization control (e.g., DapB; Advanced Cell Diagnostics)
  • Electrostatically charged slides (e.g., SuperFrost Plus; VWR Scientific)
  • 60ºC drying oven
  • Slide scanner: e.g., Hamamatsu NanoZoomer
  • Software for morphometric measurements (e.g., Hamamatsu NanoZoomer)

Support Protocol 2: Immune Response Measurement by Real‐Time Quantitative PCR (RT‐qPCR) of Bulla Fluids

  Additional Materials (also see protocol 1)
  • Germ‐free Junbo mice (MRC Harwell Institute)
  • RNase‐free H 2O
  • Nucleospin RNA/protein isolation kits (Macherey‐Nagel)
  • AB 7500 software v2.0.1
  • Additional reagents and equipment for quantitative PCR (qPCR; Bookout et al., ; Cheeseman et al., )

Basic Protocol 2: Mouse Immunization and Protection Against NTHi Infection

  Materials
  • 5‐week‐old SPF Junbo mice (MRC Harwell Institute; see protocol 1 materials list for more information)
  • Live NTHi bacteria ( protocol 1, steps 1 to 4) for intranasal challenge post‐immunization
  • Supplemented brain‐heart infusion (sBHI) broth and agar plates (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe) containing 0.08% (w/v) paraformaldehyde
  • Phosphate‐buffered saline (PBS; see recipe)
  • Adjuvant (Adjuplex from Sigma)
  • 1.1‐ml Z‐Gel spin columns (Sarstedt)
  • Microscope slides
  • Microscope with phase‐contrast optics
  • Additional reagents and equipment for blood collection from mice (Donovan and Brown, ), injection of mice (Donovan and Brown, ), and intranasal inoculation and determination of ME infection rate and bacterial titer by terminal sampling ( protocol 1)

Basic Protocol 3: Antimicrobial Treatment of NTHi Infection

  Materials
  • 8‐week old SPF Junbo mice (MRC Harwell Institute; see protocol 1 materials list for more information)
  • NTHi bacteria for intranasal challenge as described in protocol 1
  • Azithromycin (Sigma‐Aldrich, cat. no. 57947) in 2% methoxycellulose (or other antibiotic in solution as appropriate)
  • 2% methoxycellulose (Sigma‐Aldrich, cat. no. M7140)
  • Gavage needle
  • Additional reagents and equipment for intranasal inoculation and determination of ME infection rate and bacterial titer by terminal sampling ( protocol 1)

Basic Protocol 4: NTHi Mouse Pulmonary Infection Model

  Materials
  • NTHi bacteria for intranasal challenge (see protocol 1)
  • 8‐week‐old SPF Junbo mice (see protocol 1 materials list for more information)
  • Homogenizer (we use an IKA Ultra‐Turax T25 operated in a Class II microbiological safety cabinet)
  • Additional reagents and equipment for intranasal inoculation and determination of ME infection rate and bacterial titer by terminal sampling ( protocol 1) and fixation, paraffin embedding, sectioning, and staining ( protocol 2)
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Figures

Videos

Literature Cited

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